CN112526022A - Method for detecting breast milk oligosaccharide in milk - Google Patents
Method for detecting breast milk oligosaccharide in milk Download PDFInfo
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Abstract
The invention provides a method for detecting breast milk oligosaccharide in milk, which comprises the following steps: A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution; B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected; C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk. The method adopts the precipitant to precipitate the protein, then adopts the ion exchange chromatography to measure the breast milk oligosaccharide after filtration and purification treatment, and has the advantages of simple and rapid pretreatment method, accurate quantification and stable and reliable result.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for detecting breast milk oligosaccharide in milk.
Background
Human Milk Oligosaccharides (HMOs) are the third largest solid component in human breast milk, second only to lactose and fat, and have important biological functions, not only playing an anti-infective role for intestinal pathogenic microorganisms, but also maintaining the balance of intestinal microecology. FDA and EFSA have now approved 2 '-FL and LNnT for use in infant formula, and new zealand is expected to approve 2' -FL alone or in combination with LNnT in infant formula in 2020, and regulations are also being actively promoted domestically. The development of the HMOs-containing infant formula powder and the quality control need to establish an accurate quantitative detection method of breast milk oligosaccharide in the milk powder.
At present, the HMOs quantitative determination method comprises a high performance liquid chromatography, an ultra high performance liquid chromatography-fluorescence detection method (UHPLC-FLD), a liquid chromatography-mass spectrum/mass spectrum (LC-MS/MS), a porous graphitized carbon-liquid chromatography-mass spectrum/mass spectrum (PCG-LC-MS/MS), capillary electrophoresis-electrospray ionization-mass spectrum/mass spectrum (CE-ESI-MS/MS), a high performance anion exchange chromatography-pulse electrochemical detection method (HPAEC-PAD) and the like, but most of the target samples determined by the methods are breast milk, and the detection method of breast milk oligosaccharides in the milk powder is less reported.
In the prior art, ultra-high performance liquid chromatography and mass spectrometry are adopted in the method for rapidly, qualitatively and quantitatively determining breast milk oligosaccharide, and a liquid chromatography-mass spectrometry method is adopted in the method for detecting oligosaccharide in breast milk. The addition amount of HMOs in the milk powder is possibly lower than that of HMOs in breast milk, the dilution of HMOs in the milk powder is different from that of breast milk, and the interference of lactose in the milk powder is more obvious than that in the breast milk. Therefore, the detection method of HMOs in breast milk is not suitable for milk powder, and needs to be tested and optimized. The existing detection method is low in accuracy, the content of HMOs in the infant formula powder cannot be accurately detected, and the addition amount of the HMOs in the infant formula powder cannot be controlled, so that whether the produced product meets the requirements of regulations or not cannot be judged.
Disclosure of Invention
In view of this, the technical problem to be solved by the present invention is to provide a method for detecting breast milk oligosaccharides in milk, which is simple, accurate, stable and reliable in result.
The invention relates to a method for detecting breast milk oligosaccharide in milk, which comprises the following steps:
A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution;
B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected;
C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk.
Preferably, the breast milk oligosaccharide is one or more of 3 '-sialyllactose, 6' -sialyllactose and lacto-N-tetraose.
Preferably, the milk sample comprises liquid milk or a milk powder solution obtained by dissolving milk powder in water.
Preferably, the precipitating agent in the step A) is an acid solution or an organic solvent; the acid solution is a sulfosalicylic acid solution or an acetic acid solution, and the organic solvent is ethanol or acetonitrile; preferably, the precipitating agent is a sulfosalicylic acid solution; more preferably, the concentration of the sulfosalicylic acid solution is 1-5 wt%.
Preferably, the mass ratio of the milk sample and the precipitant in the step A) is 1: 1-1: 2.
Preferably, ultrasonic extraction or oscillation extraction is adopted before the filtration in the step A), and the time for ultrasonic extraction or oscillation extraction is preferably 5-15 min.
Preferably, the filter membrane of step B) is a 0.45 μm filter membrane; the purifying column is a Dionex OnGuard II RP purifying column; the specification of the purification column is 1mL or 2.5 mL; the purifying column is activated by sequentially adopting methanol and water.
Preferably, the column of anion exchange chromatography in step C) is a DIONEX CarboPacTM PA1 column, a Dionex CarboPac PA 20; the detector is an electrochemical detector; the flow rate of the mobile phase is 0.8-1.0 mL/min, the column temperature is 30-35 ℃, and the sample injection volume is 10-50 mu L.
Preferably, the mobile phase of the anion exchange chromatography in the step C) is A deionized water, B is NaOH solution, C is NaOAc and NaOH mixed solution, and D is sodium acetate solution.
Preferably, the mobile phase B is 200-250 mmol/L NaOH solution; c is 1.0mol/L NaAc and 100mmol/L NaaOH solution, and D is NaAc solution of 90-100 mmol/L.
Preferably, the anion exchange chromatography is gradient elution, in particular:
the first stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 10-15 minutes; the second stage comprises 10-20% of mobile phase A, 40-50% of mobile phase B, 0% of mobile phase C and 40-50% of mobile phase D, and the elution time is 10-45 minutes; in the third stage, 10-15% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 40-45% of mobile phase D are adopted, and the elution time is 5-10 minutes; the fourth stage comprises 0% of mobile phase A, 0% of mobile phase B, 90% -100% of mobile phase C and 0% -10% of mobile phase D, and the elution time is 5-10 minutes; the fifth stage comprises 10-20% of mobile phase A, 70-80% of mobile phase B, 0% of mobile phase C and 0% of mobile phase D, and the elution time is 5-10 minutes; the sixth stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 5-10 minutes.
Preferably, the anion exchange chromatography is gradient elution, in particular:
58% of mobile phase A, 40% of mobile phase B, 0% of mobile phase C and 2% of mobile phase D in 0-10 min;
58% of mobile phase A in 10-40 min → 15%; mobile phase B40%, mobile phase C0% and mobile phase D2% → 45%;
the mobile phase A accounts for 15% in 40-45 min; mobile phase B40%, mobile phase C0% and mobile phase D45%;
45-45.1 min mobile phase A is 15% → 0%; mobile phase B40% → 0%, mobile phase C0% → 100% and mobile phase D45% → 0%;
the mobile phase A is 0% in 45.1-55 min; mobile phase B is 0%, mobile phase C is 100% and mobile phase D is 0%;
55-55.1 min mobile phase A is 0% → 20%; mobile phase B0% → 80%, mobile phase C100% → 0% and mobile phase D0%;
the mobile phase A accounts for 20% in 55.1-60 min; 80% for mobile phase B, 0% for mobile phase C and 0% for mobile phase D;
20% → 58% of mobile phase A in 60-60.1 min; mobile phase B80% → 40%, mobile phase C0% and mobile phase D0% → 2%;
58% of mobile phase A in 60.1-65 min; mobile phase B was 40%, mobile phase C was 0% and mobile phase D was 2%.
Preferably, the injection concentration of the 3' -sialyllactose is 0.20 mu g/mL-20.0 mu g/mL; the sample injection concentration of the 6' -sialyllactose is 0.20 mu g/mL-20.0 mu g/mL; the sample injection concentration of the lactose-N-tetrasaccharide is 0.20 mu g/mL-20.0 mu g/mL.
Compared with the prior art, the invention provides a method for detecting breast milk oligosaccharide in milk, which comprises the following steps: A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution; B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected; C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk. The method adopts the precipitant to precipitate the protein, then adopts the ion exchange chromatography to measure the breast milk oligosaccharide after filtration and purification treatment, and has the advantages of simple and rapid pretreatment method, accurate quantification and stable and reliable result.
Drawings
FIG. 1 is a chromatogram of a 3' -SL standard;
FIG. 2 is a chromatogram of a 6' -SL standard;
FIG. 3 is a chromatogram of an LNT standard;
FIG. 4 is a chromatogram of a milk powder sample added with 3' -SL;
FIG. 5 is a chromatogram of a milk powder sample added with 6' -SL;
FIG. 6 is a chromatogram of a milk powder sample supplemented with LNT;
FIG. 7 is a chromatogram of a milk powder sample supplemented with 3-FL;
FIG. 8 is a 3' -SL labeling map in comparative example 2;
FIG. 9 is the 6' -SL labeling map of comparative example 2;
FIG. 10 is an LNT spiking map of comparative example 2.
Detailed Description
The invention provides a method for detecting breast milk oligosaccharide in milk, and a person skilled in the art can use the contents to reference the contents and appropriately improve process parameters to realize the detection. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the scope of the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the description of the present invention, reference to "one embodiment" means that a particular feature, structure, or parameter, step, or the like described in the embodiment is included in at least one embodiment according to the present invention. Thus, appearances of the phrases such as "in one embodiment," "in one embodiment," and the like in this specification are not necessarily all referring to the same embodiment, nor are other phrases such as "in another embodiment," "in a different embodiment," and the like. Those of skill in the art will understand that the particular features, structures or parameters, steps, etc., disclosed in one or more embodiments of the present description may be combined in any suitable manner.
The invention relates to a method for detecting breast milk oligosaccharide in milk, which comprises the following steps:
A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution;
B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected;
C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk.
The detection method of the breast milk oligosaccharide in milk provided by the invention is particularly suitable for detecting the breast milk oligosaccharide in milk powder, in particular to one or more of 3 '-sialyllactose, 6' -sialyllactose and lactose-N-tetraose.
In some embodiments, the method includes the steps of pre-treating the milk powder and qualitatively and quantitatively analyzing the breast milk oligosaccharides of 3 '-sialyllactose, 6' -sialyllactose, lacto-N-tetraose in the pre-treated milk powder. The invention improves the pretreatment operation of the sample, and overcomes the defects of complex pretreatment, high cost, long time and the like in the prior art; the invention adopts an ion chromatographic system containing an anion exchange chromatographic column, and well distinguishes breast milk oligosaccharides such as 3 '-sialyllactose, 6' -sialyllactose, lactose-N-tetraose and the like from other similar substances by selecting proper chromatographic conditions, thereby improving the accuracy of detection results.
The method for detecting the breast milk oligosaccharide in milk provided by the invention comprises the steps of firstly precipitating protein in a milk sample by using a precipitator, and filtering to obtain a sample solution. Preferably, the milk sample is mixed with water, precipitated with precipitant, ultrasonically or vibrationally extracted, and filtered.
The milk sample of the present invention includes liquid milk or a milk powder solution obtained by dissolving milk powder in water. Wherein the liquid milk sample can be breast milk, cow milk, goat milk, etc.
According to the invention, the mixing ratio of the milk sample and water is preferably 0.1-0.5 g: 10-25 mL; more preferably 0.2 to 0.4 g: 15-23 mL; the mixing method of the present invention is not limited, and those skilled in the art will be familiar with it.
The precipitating agent of the invention is preferably an acid solution or an organic solvent; more preferably an acid solution. The acid solution is a sulfosalicylic acid solution or an acetic acid solution, and the organic solvent is ethanol or acetonitrile; most preferably, the precipitating agent is a sulfosalicylic acid solution; particularly preferably, the concentration of the sulfosalicylic acid solution is 1-5 wt%; specifically, it may be 2 wt%, 3 wt% or 4 wt%
The mass ratio of the milk sample to the precipitator is 1: 1-1: 2; preferably 1:1.5.
The time for ultrasonic extraction is preferably 5-15 min; specifically, the time period can be 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min or 14 min. The frequency of the ultrasonic extraction is not limited in the present invention, and is well known to those skilled in the art.
The filtration is not limited in the present invention, and may be filtration using filter paper.
And purifying the sample solution by adopting a filter membrane and a purification column to obtain the solution to be detected. The sample solution passes through a 0.45 mu m filter membrane and a purification column in turn, the filtrate with 2 times of the column volume in the front is discarded, and the subsequent filtrate is collected and is measured on a computer. Appropriate dilution may be performed as necessary.
The filter membrane of the invention is preferably a 0.45 μm filter membrane; the purifying column is preferably a Dionex OnGuard II RP purifying column; the specification of the purification column is 1mL or 2.5 mL; the sample purification step includes a step of activating the purification column, and for example, the purification column may be activated with methanol and water in this order and used after being left for half an hour. The dosage of the methanol can be 5-15 ml, and the dosage of the water can be 10-20 ml.
And (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk.
In the sample detection step, 3 ' -SL (3 ' -sialyllactose, 6 ' -sialyllactose, lacto-N-tetraose) and other breast milk oligosaccharide standard substances are weighed, dissolved by water and subjected to constant volume to serve as stock solutions. And (4) absorbing different amounts of stock solutions for dilution, and preparing a series of standard working solutions with concentrations. And (3) measuring the standard working solution and the sample measuring solution by adopting an ion chromatograph, and quantifying by adopting an external standard method and combining the peak area percentage.
The type and specification of the ion exchange chromatograph are not limited in the present invention, and those skilled in the art are familiar with the present invention. The ion chromatograph is equipped with an electrochemical detector, and the anion exchange chromatography column of the invention is preferably a DIONEX CarboPacTM PA1 column or a Dionex CarboPac PA20 column.
Wherein the flow rate of the mobile phase is preferably 0.4-0.8 mL/min, and more preferably 0.4-0.45 mL/min; the column temperature is preferably 30-35 ℃, and more preferably 30 ℃; the sampling volume is preferably 10-50 mu L; more preferably 10 to 30. mu.L.
The anion exchange chromatography is gradient elution, wherein the mobile phase of the anion exchange chromatography is A deionized water, B is NaOH solution, C is NaAc and NaOH mixed solution, and D is NaAc solution.
Specifically, the mobile phase B is 200-250 mmol/L NaOH solution; c is 1.0mol/L NaAc and 100mmol/L NaaOH solution, and D is NaAc solution of 90-100 mmol/L.
In some preferred embodiments of the present invention, the gradient elution is specifically:
the first stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 10-15 minutes; the second stage comprises 10-20% of mobile phase A, 40-50% of mobile phase B, 0% of mobile phase C and 40-50% of mobile phase D, and the elution time is 10-45 minutes; in the third stage, 10-15% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 40-45% of mobile phase D are adopted, and the elution time is 5-10 minutes; the fourth stage comprises 0% of mobile phase A, 0% of mobile phase B, 90% -100% of mobile phase C and 0% -10% of mobile phase D, and the elution time is 5-10 minutes; the fifth stage comprises 10-20% of mobile phase A, 70-80% of mobile phase B, 0% of mobile phase C and 0% of mobile phase D, and the elution time is 5-10 minutes; the sixth stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 5-10 minutes.
In some preferred embodiments of the present invention, the gradient elution is specifically:
58% of mobile phase A, 40% of mobile phase B, 0% of mobile phase C and 2% of mobile phase D in 0-10 min;
58% of mobile phase A in 10-40 min → 15%; mobile phase B40%, mobile phase C0% and mobile phase D2% → 45%;
the mobile phase A accounts for 15% in 40-45 min; mobile phase B40%, mobile phase C0% and mobile phase D45%;
45-45.1 min mobile phase A is 15% → 0%; mobile phase B40% → 0%, mobile phase C0% → 100% and mobile phase D45% → 0%;
the mobile phase A is 0% in 45.1-55 min; mobile phase B is 0%, mobile phase C is 100% and mobile phase D is 0%;
55-55.1 min mobile phase A is 0% → 20%; mobile phase B0% → 80%, mobile phase C100% → 0% and mobile phase D0%;
the mobile phase A accounts for 20% in 55.1-60 min; 80% for mobile phase B, 0% for mobile phase C and 0% for mobile phase D;
20% → 58% of mobile phase A in 60-60.1 min; mobile phase B80% → 40%, mobile phase C0% and mobile phase D0% → 2%;
58% of mobile phase A in 60.1-65 min; mobile phase B was 40%, mobile phase C was 0% and mobile phase D was 2%.
The sample injection concentration of the 3' -sialyllactose is 0.20 mu g/mL-20.0 mu g/mL; the sample injection concentration of the 6' -sialyllactose is 0.20 mu g/mL-20.0 mu g/mL; the sample injection concentration of the lactose-N-tetrasaccharide is 0.20 mu g/mL-20.0 mu g/mL. Within the above range, a good linear relationship can be exhibited.
The invention provides a method for detecting breast milk oligosaccharide in milk, which comprises the following steps: A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution; B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected; C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk. The method adopts the precipitant to precipitate the protein, then adopts the ion exchange chromatography to measure the breast milk oligosaccharide after filtration and purification treatment, and has the advantages of simple and rapid pretreatment method, accurate quantification and stable and reliable result.
In order to further illustrate the present invention, the following will describe the method for detecting breast milk oligosaccharides in milk according to the present invention in detail with reference to the following examples.
EXAMPLE 1 determination of the content of 3 '-SL, 6' -SL and LNT in the formula
1. Sample pretreatment: weighing 0.1g of milk powder sample in a 25mL colorimetric tube, adding 10mL of deionized water, dissolving and uniformly mixing, adding 5mL of 3 wt% sulfosalicylic acid solution, adding water to a constant volume, shaking up after the constant volume is scaled, and performing ultrasonic extraction for 10 min. Shaking up, filtering with qualitative filter paper.
2. Purifying: the activated purification column was activated with 10mL of methanol and 15mL of water in this order before use, and was left to stand for half an hour before use.
The sample solution passes through a 0.45 mu m filter membrane and a purification column in turn, the filtrate with 2 times of the column volume in the front is discarded, and the subsequent filtrate is collected and is measured on a computer.
3. Measuring by an instrument:
and (3) measuring the sample and the standard working solution according to the ion chromatography condition, correcting the concentration of the sample solution by using a standard curve, and quantifying by adopting an external standard method.
Ion chromatographic column: a Dionex CarboPac PA20 anion exchange column;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
leacheate: a is deionized water, B is 250mM NaOH aqueous solution, C is 1.0mol/L NaAc &100mmol/L LNaOH, D is 100mmol/L NaAc solution, and the gradient elution conditions are shown in Table 1.
TABLE 1 mobile phase gradient elution conditions
4. Linear range
Accurately absorbing the 3 '-SL, 6' -SL and LNT standard solutions, diluting with water to prepare standard working solutions with the concentrations of 0.20 mu g/mL, 0.40 mu g/mL, 1.0 mu g/mL, 2.0 mu g/mL, 4.0 mu g/mL, 8.0 mu g/mL and 20.0 mu g/mL, drawing a standard curve by taking the peak area of the working solution as a horizontal ordinate and the concentration of the corresponding working solution as a vertical ordinate to obtain a regression equation, wherein the correlation coefficients are all larger than 0.990. The 3 '-SL, 6' -SL and LNT standard chromatograms are shown in FIGS. 1-3, respectively. FIG. 1 is a chromatogram of a 3' -SL standard; FIG. 2 is a chromatogram of a 6' -SL standard; FIG. 3 is a chromatogram of an LNT standard.
5. Recovery rate of added standard
The results of the recovery rate after the addition of the standard in the blank milk powder are shown in Table 2. The chromatogram of the added 3' -SL milk powder sample is shown in figure 4; the chromatogram of the added 6' -SL milk powder sample is shown in figure 5; the sample chromatogram of LNT-added milk powder is shown in FIG. 6.
TABLE 2 recovery with addition of standard
Comparative example 1 determination of the content of 3-FL in the formula milk powder
1. Sample pretreatment: weighing 0.1g of milk powder sample in a 25mL colorimetric tube, adding 10mL of deionized water, dissolving and uniformly mixing, adding 5mL of 3 wt% sulfosalicylic acid solution, adding water to a constant volume, shaking up after the constant volume is scaled, and performing ultrasonic extraction for 10 min. Shaking up, filtering with qualitative filter paper.
2. Purifying: the activated purification column was activated with 10mL of methanol and 15mL of water in this order before use, and was left to stand for half an hour before use.
The sample solution passes through a 0.45 mu m filter membrane and a purification column in turn, the filtrate with 2 times of the column volume in the front is discarded, and the subsequent filtrate is collected and is measured on a computer.
3. Measuring by an instrument:
and (3) measuring the sample and the standard working solution according to the ion chromatography condition, correcting the concentration of the sample solution by using a standard curve, and quantifying by adopting an external standard method.
Ion chromatographic column: a Dionex CarboPac PA20 anion exchange column;
column temperature: 30 ℃;
sample introduction amount: 10 mu L of the solution;
leacheate: a is deionized water, B is 250mM NaOH aqueous solution, C is 1.0mol/LNaOAc and 100mmol/LNaOH, D is 100mmol/L sodium acetate solution, and gradient elution conditions are shown in Table 3.
TABLE 3 mobile phase gradient elution conditions
4. The chromatogram of the milk powder sample added with 3-FL is shown in figure 7. 3-FL peak time is fast, in the sample is completely covered by lactose peak, can not be quantitative.
Comparative example 2 determination of the content of 3 '-SL, 6' -SL and LNT in the formula
1. Sample pretreatment
Weighing 2.5g of milk powder sample of certain mass, dissolving in 50mL of 0.15% acetic acid solution in advance, centrifuging at 10000rpm for 15min, collecting intermediate clear liquid, and passing through 0.22 μm nylonFiltering with microporous membrane, collecting supernatant, diluting with ultrapure water (more than 20 times), and passing through SPE-C18And (5) carrying out column and finally carrying out machine test.
2. Quantitative detection by high performance anion exchange chromatography (with pulsed amperometric detector):
detecting with an ampere detector by adopting a four-potential pulse ampere method, wherein a reference electrode is Ag/AgCl, a Dionex CarboPac PA20 anion exchange column, and gradient elution and leaching conditions are adopted: gradient elution was performed using 0.25M NaOH and 1M NAC as eluents at a flow rate of 0.5 mL/min. Column temperature: at 30 ℃. Sample introduction volume: 10 μ L. The analysis conditions were as follows:
0-15min,10％A+90％C；
15-25min,20％A+80％C；
25-27min,30～24％A+0～36％B+70～40％C；
27～35min,24％A+36％B+40％C；
35-37min,24～10％A+36～0％B+40～90％C；
37～50min,10％A+90％C。
3. adding a standard:
(1) 2 parts of the sample (blank milk powder sample without 3 '-SL) which is treated in the same way are taken, and 300mg/100g of quantitative 3' -SL standard substance is added; 2 portions were simultaneously analyzed according to the same analytical procedure. The map is shown in FIG. 8. As can be seen from fig. 8, the target peak is severely disturbed by the surrounding clutter.
(2) 2 parts of the sample (blank milk powder sample without 6 '-SL) which is treated in the same way are taken, and 300mg/100g of quantitative 6' -SL standard substance is added; 2 portions were simultaneously analyzed according to the same analytical procedure. The map is shown in FIG. 9. As can be seen from FIG. 9, the separation of 6' -SL is poor.
(3) Taking 2 parts of the sample (blank milk powder sample without LNT) subjected to the same treatment, and adding 300mg/100g of quantitative LNT standard; 2 portions were simultaneously analyzed according to the same analytical procedure. The map is shown in FIG. 10. As can be seen from fig. 10, the target peak is severely interfered by the surrounding peaks and cannot be separated.
Comparing the above examples and comparative examples, it can be seen that the method of the present invention has simple sample pretreatment operation, high detection accuracy, and can accurately quantify the 3 '-SL, 6' -SL, LNT content in milk, and the 3-FL content in milk cannot be detected.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (13)
1. A method for detecting breast milk oligosaccharide in milk comprises the following steps:
A) precipitating protein in a milk sample by adopting a precipitator, and filtering to obtain a sample solution;
B) purifying the sample solution by adopting a filter membrane and a purification column to obtain a solution to be detected;
C) and (3) determining the liquid to be detected by adopting anion exchange chromatography to obtain the content of the breast milk oligosaccharide in the milk.
2. The assay of claim 1, wherein the breast milk oligosaccharide is one or more of 3 '-sialyllactose, 6' -sialyllactose and lacto-N-tetraose.
3. The detection method according to claim 1, wherein the milk sample comprises liquid milk or a milk powder solution obtained by dissolving milk powder in water.
4. The detection method according to claim 1, wherein the precipitant in step a) is an acid solution or an organic solvent; the acid solution is a sulfosalicylic acid solution or an acetic acid solution, and the organic solvent is ethanol or acetonitrile; preferably, the precipitating agent is a sulfosalicylic acid solution; more preferably, the concentration of the sulfosalicylic acid solution is 1-5 wt%.
5. The detection method according to claim 1, wherein the mass ratio of the milk sample and the precipitant in step A) is 1:1 to 1: 2.
6. The detection method according to claim 1, wherein the filtering in step a) further comprises ultrasonic extraction or oscillation extraction, and preferably, the time for ultrasonic extraction or oscillation extraction is 5-15 min.
7. The detection method according to claim 1, wherein the filter in step B) is a 0.45 μm filter; the purifying column is a Dionex OnGuard II RP purifying column; the specification of the purification column is 1mL or 2.5 mL; the purifying column is activated by sequentially adopting methanol and water.
8. The detection method as claimed in claim 1, wherein the anion exchange chromatography column of step C) is a DIONEX CarboPac PA1 column or DIONEX CarboPac PA 20; the detector is an electrochemical detector; the flow rate of the mobile phase is 0.8-1.0 mL/min, the column temperature is 30-35 ℃, and the sample injection volume is 10-50 mu L.
9. The detection method according to claim 1, wherein the mobile phase of the anion exchange chromatography in the step C) is A which is deionized water, B which is NaOH solution, C which is NaAc and NaOH mixed solution, and D which is NaAc solution.
10. The detection method according to claim 9, wherein the mobile phase B is 200-250 mmol/L NaOH solution; c is 1.0mol/L NaAc and 100mmol/L NaaOH solution, and D is NaAc solution of 90-100 mmol/L.
11. The detection method according to claim 10, characterized in that the anion exchange chromatography is a gradient elution, in particular:
the first stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 10-15 minutes; the second stage comprises 10-20% of mobile phase A, 40-50% of mobile phase B, 0% of mobile phase C and 40-50% of mobile phase D, and the elution time is 10-45 minutes; in the third stage, 10-15% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 40-45% of mobile phase D are adopted, and the elution time is 5-10 minutes; the fourth stage comprises 0% of mobile phase A, 0% of mobile phase B, 90% -100% of mobile phase C and 0% -10% of mobile phase D, and the elution time is 5-10 minutes; the fifth stage comprises 10-20% of mobile phase A, 70-80% of mobile phase B, 0% of mobile phase C and 0% of mobile phase D, and the elution time is 5-10 minutes; the sixth stage comprises 50-60% of mobile phase A, 30-40% of mobile phase B, 0% of mobile phase C and 2-5% of mobile phase D, and the elution is carried out for 5-10 minutes.
12. The detection method according to claim 11, characterized in that the anion exchange chromatography is a gradient elution, in particular:
58% of mobile phase A, 40% of mobile phase B, 0% of mobile phase C and 2% of mobile phase D in 0-10 min;
58% of mobile phase A in 10-40 min → 15%; mobile phase B40%, mobile phase C0% and mobile phase D2% → 45%;
the mobile phase A accounts for 15% in 40-45 min; mobile phase B40%, mobile phase C0% and mobile phase D45%;
45-45.1 min mobile phase A is 15% → 0%; mobile phase B40% → 0%, mobile phase C0% → 100% and mobile phase D45% → 0%;
the mobile phase A is 0% in 45.1-55 min; mobile phase B is 0%, mobile phase C is 100% and mobile phase D is 0%;
55-55.1 min mobile phase A is 0% → 20%; mobile phase B0% → 80%, mobile phase C100% → 0% and mobile phase D0%;
the mobile phase A accounts for 20% in 55.1-60 min; 80% for mobile phase B, 0% for mobile phase C and 0% for mobile phase D;
20% → 58% of mobile phase A in 60-60.1 min; mobile phase B80% → 40%, mobile phase C0% and mobile phase D0% → 2%;
58% of mobile phase A in 60.1-65 min; mobile phase B was 40%, mobile phase C was 0% and mobile phase D was 2%.
13. The detection method according to claim 2, wherein the injection concentration of the 3' -sialyllactose is from 0.20 μ g/mL to 20.0 μ g/mL; the sample injection concentration of the 6' -sialyllactose is 0.20 mu g/mL-20.0 mu g/mL; the sample injection concentration of the lactose-N-tetrasaccharide is 0.20 mu g/mL-20.0 mu g/mL.
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